Atractylenolide III ameliorates spinal cord injury in rats by modulating microglial/macrophage polarization

Abstract Background Inflammatory reactions induced by spinal cord injury (SCI) are essential for recovery after SCI. Atractylenolide III (ATL‐III) is a natural monomeric herbal bioactive compound that is mainly derived in Atractylodes macrocephala Koidz and has anti‐inflammatory and neuroprotective effects. Objective Here, we speculated that ATL‐III may ameliorate SCI by modulating microglial/macrophage polarization. In the present research, we focused on investigating the role of ATL‐III on SCI in rats and explored the potential mechanism. Methods The protective and anti‐inflammatory effects of ATL‐III on neuronal cells were examined in a rat SCI model and lipopolysaccharide (LPS)‐stimulated BV2 microglial line. The spinal cord lesion area, myelin integrity, and surviving neurons were assessed by specific staining. Locomotor function was evaluated by the Basso, Beattie, and Bresnahan (BBB) scale, grid walk test, and footprint test. The activation and polarization of microglia/macrophages were assessed by immunohistofluorescence and flow cytometry. The expression of corresponding inflammatory factors from M1/M2 and the activation of relevant signaling pathways were assessed by Western blotting. Results ATL‐III effectively improved histological and functional recovery in SCI rats. Furthermore, ATL‐III promoted the transformation of M1 into M2 and attenuated the activation of microglia/macrophages, further suppressing the expression of corresponding inflammatory mediators. This effect may be partly mediated by inhibition of neuroinflammation through the NF‐κB, JNK MAPK, p38 MAPK, and Akt pathways. Conclusion This study reveals a novel effect of ATL‐III in the regulation of microglial/macrophage polarization and provides initial evidence that ATL‐III has potential therapeutic benefits in SCI rats.


| INTRODUC TI ON
Spinal cord injury (SCI) is caused by various factors and leads to sensory and motor dysfunction and eventually varying degrees of paralysis. 1 The process of SCI includes two stages: primary injury induced by the initial impact and secondary injury resulting from inflammatory environmental factors. 2 Although primary injury cannot be avoided, the biochemical and cellular events in the secondary injury stage are essential for recovery after SCI. 3 Thus, the current clinical treatment strategies for SCI mainly involve ameliorating secondary injury, and identifying drugs that can effectively alleviate secondary injury is important.
In recent years, a large number of reports have confirmed that Chinese herbal medicines, especially natural monomers derived from plant extracts, effectively treat inflammation and immune-related diseases with lower toxicity and drug resistance. 4 Atractylenolide III (ATL-III) is the main bioactive constituent of Atractylodes macrocephala Koidz and has a wide range of sources, such as Codonopsis pilosula and Chloranthus henryi Hemsl. 5 According to reports, ATL-III exhibits a series of pharmacological activities, including the inhibition of inflammation, antitumor and antiallergic responses, antagonizing apoptosis and neuroprotection. [5][6][7] Wang et al 8  showed that ATL-III has an obvious neuroprotective effect against neuronal apoptosis induced by glutamate and may have therapeutic potential for nervous system diseases mediated by excitotoxicity.
Furthermore, it was also reported that ATL-III can significantly alleviate learning and memory dysfunction caused by long-term administration of high-dose homocysteine in animals and prevent apoptosis of primary cultured neurons induced by homocysteine. 7 Recent studies have found that ATL-III can alleviate cerebral ischemic injury by reducing neuroinflammation both in vivo and in vitro. 10 Nevertheless, the role of ATL-III in SCI remains unknown. This study examined the effect of ATL-III in ameliorating SCI and explored the underlying molecular mechanisms in vivo and in vitro, providing new insights into SCI treatment.
All animal husbandry procedures were executed according to the Administration of Affairs Concerning Experimental Animals Guidelines (Ministry of Science and Technology;2004), and all experiments were performed according to ARRIVE guidelines. 11

| Contusive SCI and drug administration
Seventy-two rats were randomly divided into 3 groups: a sham group (Sham), a control group (Ctrl), and an ATL-III treatment group (ATL-III). Contusion SCI was established with an Infinite Horizon impactor (Precision Systems). 12 In brief, the anaesthetization of rat was executed with pentobarbital (50 mg/kg, i.p.), and a T9 vertebral laminectomy was performed. Moderate SCI was established with a 2.5-mm-diameter rod (force 120 kdynes). The sham rats underwent the same surgical procedure without contusion injury. Postoperatively, the rats were placed in a room with controlled conditions (20 ± 2°C, 55 ± 10% humidity). To protect against infection, the rats were administered 50 mg/kg chloramphenicol (Sangon Biotech) by drinking water. ATL-III (Phytomarker Ltd.) was mixed with 0.9% NaCl and dimethyl sulfoxide (DMSO, 1%). The rats were administered 5 mg/kg ATL-III by gavage three hours after the operation and then once a day until sacrifice. [6][7][8] The control rats were administered the same volume of saline.

| Behavioral assessments
Animals that survived for more than 42 days underwent the grid walking test, the open field test, and footprint analysis as previously described. [13][14][15] The behavioral tests were performed by 2 trained investigators who were blinded to the animal grouping information.
An open field and the Basso, Beattie, and Bresnahan (BBB) locomotor scale were used to evaluate locomotor recovery at specific time points. The crawling ability of the rats was scored on a scale of 0 to 21 over 4 min.
The grid walking test was carried out using a 100 × 100 cm grid runway with 50 × 50 mm holes formed by the rails of the grid. The rats were allowed to walk freely on the grid runway for 4 min each trial. At least 2 min of continuous walking time was required for analysis. When the paws and heel of the hind limb fell completely below the grid, an error was scored. Two investigators counted the total number of footfalls for one hind limb and the number of errors and calculated the mean error rate. Footprint analysis was performed by evaluating the forepaw and hindpaw prints made by rats with red and blue ink as they passed through a narrow runway. To assess the weight support and gait of the rats, a 4-point scoring system was used as described previously: 0, persistent limping or hind leg dragging, inability to make visible footprints; 1, visible toe prints for at least 3 toes in at least 3 footprints; 2, footprint showing exo-or endo-rotation more than double the baseline angle; 3, no obvious dragging, only exo-or endorotation; 4, no rotation.

| Tissue preparation
At predetermined time points after SCI, the rats were anesthetized.
The hearts of the rats were perfused with phosphate-buffered saline (PBS) and then with 4% paraformaldehyde (PFA). After perfusion, a 10-mm segment of the spinal cord that included the injury epicenter was removed (or the same spinal segments for sham rats), postfixed in 4% PFA, and then frozen in OCT (TissueTek, Miles, Elkart, IN).

| Histological analysis
At 42 days postinjury, tissue from the remaining rats was obtained and sectioned as described in the "Tissue preparation" section. Luxol fast blue (LFB), hematoxylin-eosin (HE) and Nissl staining (Beyotime Biotechnology, China) were performed according to the manufacturer's instructions to assess the damaged area, the medullary white matter area and the number of remaining motor neurons as previously described. 16 Nine cross-sectional images were collected from the lesion epicenter (the section containing the largest lesion area and the least sparing of white matter), and light microscopy was used to visualize the sections 1, 2, 3, and 4 mm rostral and caudal to the epicenter. The lesion area and medullary white matter area were measured using ImageJ software (National Institutes of Health) and Image-Pro 5.1 (Media Cybernetics), respectively, and normalized to the percentage of the entire stained area in an unbiased stereological manner for quantitative analysis. 16 Surviving neurons were quantified by counting the number of Nissl bodies.

| Cell viability assay
The Cell Counting Kit-8 (CCK-8) assay (Vazyme Biotech) was used to evaluate the viability of BV2 cells in the ATL-III (1, 10, or 100 µM) and vehicle groups. Briefly, when the BV2 cells reached 60% confluence, the culture medium was replaced with fresh medium containing different doses of ATL-III (1, 10, or 100 µM), and the cells were incubated for 24 h. The cells were then cultured with fresh medium containing CCK-8 reagent for 2 h. Finally, the absorbance was measured at 450 nm with a microplate reader (Thermo Fisher Scientific).

| Immunohistofluorescence
At 7 days postinjury, the spinal cords of the rats were extracted to prepare frozen sections as described above. For immunohistochemical staining, the sections were thawed at 37°C, washed with PBS, and blocked with 1% serum (Sigma-Aldrich) at 37°C for 1 h. The sections were incubated with antibodies ( Table 1) for 12 h at 4°C. The next day, the sections were washed with PBS and then incubated with secondary antibodies at 37°C for 1 h. Finally, a ZEISS Axio observation microscope with Zen imaging software (Carl Zeiss AG) was used to acquire and analyze images. The cells were counted as described in a previous report. 18

| Flow cytometry
To obtain spinal cord tissue, rats were anesthetized 7 days following surgery and then perfused with PBS. A 10-mm segment of the spinal cord, including the injury epicenter, was removed and dissociated into a single-cell suspension by sufficient trituration as previously described. 19 To analyze the proportion of microglial subtypes, the cell suspension was incubated with antibodies ( Table 1)

| Western blot analysis
Ten-millimeter spinal cord segments were extracted from rats euthanized as described above at 7 days postinjury. Protein was extracted from the spinal cord segments using radioimmunoprecipitation (RIPA) lysis buffer (Beyotime Biotechnology) containing phosphatase inhibitor cocktail (1:50; Beyotime Biotechnology) and protease inhibitors (1:100; Thermo Fisher Scientific) after they were fully homogenized. A bicinchoninic acid protein assay kit (Beyotime Biotechnology) was employed to quantify the protein concentration. The Western blot membranes were incubated with primary antibodies ( Table 1) at 4°C overnight. After that, the membranes were washed with Tris-buffered saline-Tween-20 (TBST, Biosharp) and incubated with secondary antibody at 37°C for 2 h. After incubation, the membranes were washed against with TBST three times at 37°C for 10 min, and a chemiluminescence kit (Thermo Fisher Scientific) was employed to visualize the immunoreactivity of the target proteins. The data were acquired and quantified using a ChemiDoc XRS imaging system (Bio-Rad) and ImageJ software (Bio-Rad). The gray values of specific bands were analyzed by normalizing their levels to β-actin ( Table 1).

| Statistical analysis
AII results are presented as the mean ± SD. For two-group comparisons, Student's t test or Mann-Whitney U (nonparametric tests) was used. One-way anova or Kruskal-Wallis test (nonparametric tests) followed by the Bonferroni test was used for multiple groups.
Nonparametric tests were conducted because of the nonnormal distribution and nonhomogeneity of variance. SPSS software v.14.0 (SPSS Inc.) was employed for statistical analysis, and the differences were defined as p < 0.05.

| ATL-III ameliorated the motor deficits after SCI in rats
Motor function was assessed at designated time points after SCI by the BBB locomotor scale, the grid walk test and footprint analysis. At 14-42 days following injury, the BBB scores of the ATL-III group were obviously higher than those of the control rats (all p < 0.05; Figure 1A). The ability to control the hindlimbs was examined by the grid walk test at 28, 35 and 42 days postinjury. As shown in Figure 1(B), the error rate (rate of footfalls on the grid) in the ATL-III group was observably lower than that in the control rats (all p < 0.01). Likewise, the ATL-III group scored higher in the footprint analysis than the control group at day 42 (p < 0.05; Figure 1C,D). These data demonstrated that ATL-III administration could observably improve motor function in an SCI model.

| ATL-III attenuated spinal cord tissue damage
To assess the effects of ATL-III on histopathology following SCI, the lesion area, myelinated white matter, and remnant motor neurons were measured by HE, LFB, and Nissl staining. The images suggested TA B L E 1 that rats in the ATL-III group had an observably smaller lesion area and larger LFB-positive region at the epicenter (0) and the lateral (+) and caudal (−) sides 1 mm from the epicenter compared to those in the control rats (all p < 0.05; Figure 2A,B). The numbers of remaining motor neurons 3 and 4 mm rostral and caudal to the epicenter of the ATL-III rats were greater than those in the control rats (all p < 0.05; Figure 2C).

| ATL-III reduced the ratio of activated microglia/macrophages in SCI rats
To identify the effect of ATL-III on the status and number of microglia/ macrophages in SCI rats, we performed immunohistofluorescence (IF) and flow cytometry (FCM). CD11b is a maker of microglia/macrophages, and CD68 is a maker of activated microglia/macrophages; The data are presented as the mean ± SD (n = 6). *p < 0.05, **p < 0.01 compared to the control rats thus, CD11b + CD68 + cells are activated microglia/macrophages. 19,20 As shown in Figure 3(A), CD68 + cells were very scarce in the sham rats, while there was an observably increase in the number of these cells in the control and ATL-III rats (all p < 0.01; Figure 3D). The number of CD68 + cells was obviously lower in the ATL-III rats than in the control rats (p < 0.05; Figure 3D). As shown in Figure 3(B,C), the number of CD11b + cells was obviously increased in the control and ATL-III rats compared with the sham rats (all p < 0.01; Figure 3D).
In the sham group, typical resting microglia/macrophages had small somas and many tiny processes ( Figure 3A). Following SCI, microglia/ macrophage cells were activated and had larger and rounder somas ( Figure 3B,C). Furthermore, the number of CD11b + cells was decreased in the ATL-III rats compared with the control rats (p < 0.01; Figure 3D). As shown in Figure 3A, CD11b + CD68 + cells were virtually absent in the sham rats, and the number of these cells was obviously increased in the control and ATL-III rats (p < 0.01; Figure 3D).
Likewise, the number of CD11b + CD68 + cells was lower in the ATL-III rats than in the control rats (p < 0.05; Figure 3D).
The status and proportion of microglia/macrophages were further assessed by FCM. As shown in Figure 3(E-G), the ratios of all cells were lower in the sham rats than in the control rats (all p < 0.01; Figure 3H).
Likewise, the ratios of all cells were obviously lower in the ATL-III rats than in the control rats (all p < 0.05; Figure 3H). These results revealed that microglia/macrophages were activated and cell numbers were significantly increased following SCI and that ATL-III decreased the number and proportion of activated microglia/macrophages.

| ATL-III regulated M1/M2 differentiation of activated microglia/macrophages in SCI rats
To determine the effect of ATL-III on M1/M2 differentiation in SCI rats, we labeled M1 and M2 cells with CD68, C-C chemokine receptor type 7 (CCR7), arginase-1 (Arg-1) and CD206 and detected them via IF and FCM. 21,22 As shown in Figure 4(A-C), CD68 + CCR7 + M1 cells were rare in the sham rats, but the number of these cells was obviously increased in the control and ATL-III rats (p < 0.01; Figure 4D). There were fewer CD68 + CCR7 + M1 cells in the ATL-III rats than in the control rats (p < 0.05; Figure 4D). As shown in Figure 4(E-G), CD68 + Arg1 + M2 cells were not detected in the sham rats, but the number of these cells was significantly increased in the control and ATL-III groups (p < 0.01; Figure 4H). Remarkably, the number of CD68 + Arg1 + M2 cells in the ATL-III group was further significantly increased compared with that in the control rats (p < 0.05; Figure 4H).
To determine the effect of ATL-III on the proportion of M1/M2 differentiation in SCI rats, the panel of CD68, CCR7, and CD206 molecules was further evaluated by FCM. CD68 + CCR7 + cells were defined as M1 phenotypes, and CD68 + CD206 + cells were defined as M2 phenotypes. As shown in Figure 4(I-K), the proportion of M1 cells was observably increased in the SCI rats compared with the sham rats (p < 0.01; Figure 4L). The proportion of M1 cells was lower in the ATL-III rats than in the control rats (p < 0.05; Figure 4L). In contrast, the proportion of M2 cells was significantly higher in the ATL-III rats than in the control rats (p < 0.01; Figure 4P).

| ATL-III regulates the expression of corresponding inflammatory factors of M1/M2
To further elucidate the protective mechanism of ATL-III in SCI, the numbers of the M1/M2 cells and the level of corresponding inflammatory factors were analyzed by Western blotting. As shown in Figure 5(A,B), the level of inducible nitric oxide synthase (iNOS), which is a maker of the M1 phenotype, 23 was observably increased in control rats (p < 0.001) but was decreased in ATL-III rats (p < 0.01). The level of Arg-1 was increased in both control rats and ATL-III rats compared with sham rats (p < 0.001). The production of proinflammatory mediators, including tumor necrosis factorα (TNFα), interleukin (IL)-1β, and IL-6, was increased significantly in control rats compared with sham rats (all p < 0.01), and the production of these proinflammatory factors significantly decreased after ATL-III administration (all p < 0.05).
However, the production of the anti-inflammatory factor IL-10 by M2 macrophages was significantly decreased after SCI (p < 0.001) and increased significantly after ATL-III administration, as expected (p < 0.01). These data suggest that the protective functions of ATL-III in SCI rats and the expression of inflammatory factors are related to the regulation of the microglia/macrophage-activated phenotype.

| ATL-III regulates the M1/M2 phenotype and corresponding inflammatory factor expression after LPS stimulation in microglia
To explore whether ATL-III directly regulates the M1/M2 phenotype and the expression of corresponding inflammatory factors in microglia, BV2 microglia were stimulated with LPS to confirm the data obtained in vivo. ATL-III at doses of 1-100 μM did not obviously decrease the viability of microglia, as determined by the CCK-8 assay (all p > 0.05; Figure 6A). The Western blot results showed that the level of iNOS was decreased in ATL-III-treated microglia after LPS stimulation, whereas the level of Arg-1 was increased in response to ATL-III administration (p < 0.05; Figure 6B

| Effects of ATL-III on signaling pathways related to the polarization of microglia
To elucidate the specific mechanisms by which ATL-III regulates microglial M1/M2 polarization and exerts antineuroinflammatory effects, the activation of the NF-κB signaling pathway was analyzed by Western blotting. The levels of phosphorylated IκBα and p65, which are involved in the NF-κB signaling pathway, were significantly attenuated by ATL-III administration both in vivo and in vitro (p < 0.05; Figure 7A,B). JNK, p38, and ERK1/2 are known to activate NF-κB, which participates in the expression of multiple proinflammatory factors. [24][25][26] As shown in Figure 7(C,D), ATL-III did not affect  Figure 7E).
Likewise, we found that ATL-III activated Akt phosphorylation in microglia after LPS stimulation (p < 0.05; Figure 7F). Together, these results could explain how ATL-III could enhance the transformation from the M1 phenotype to the M2 phenotype.

| DISCUSS ION
SCI is a serious disabling disease and inflammation is the most critical pathologic change that occurs during the secondary injury phase. 27 Previous reports have proven that microglia/macrophages can be divided into two main subgroups, namely M1 (proinflammatory) microglia/macrophages and M2 (anti-inflammatory) microglia/ macrophages. 28,29 Therefore, inflammation may be ameliorated by promoting the transformation of M1 into M2. 30 However, most antiinflammatory drugs such as glucocorticoids and biological agents are not suitable for long-term use because of their side effects or drug resistance. 31 We have been greatly inspired to extract effective pharmaceutical compounds from natural plants to treat diseases. Some of these pharmaceutical compounds, such as artemisinin, which has been used for the treatment of malaria, have proven to be very successful. 32 ATL-III has multiple pharmacological properties and is virtually nontoxic even at high doses, and its high blood-brain barrier permeability makes it an ideal agent for treating neuroinflammation. 7, 33 We hypothesize that ATL-III exerts a neuroprotective effect by suppressing the activation and regulating the polarization of microglia/macrophages.
The severity of spinal cord tissue damage after SCI directly determines the extent of locomotor function recovery. 34 The results showed that BBB scores and performance on the grid walk and footprint tests were obviously improved by ATL-III administration.
After confirming that motor function recovery was improved, we evaluated pathological changes at the tissue level. The results of HE, However, our study has a few limitations. We used female rats in our study. This is because female rats are more likely than male rats to artificially urinate due to anatomical differences in the urinary system. Therefore, the use of female rats reduces infection and mortality, while also reducing the impact of non-injury factors on our experiments. It is undeniable that the sexual comparisons study may make our research more adequate. Our results show that ATL-III alleviates secondary injury after SCI by regulating the polarization of microglia; however, ATL-III may also ameliorate SCI through other means. The changes in NF-κB, MAPK, and Akt signaling may partly explain the potential mechanism underlying the neuroprotection and the anti-inflammatory effects of ATL-III, but we may have overlooked other signaling pathways. In addition, the interactions between microglia/macrophages and other glial cells, such as oligodendrocytes that make up the myelin sheath, have not been exhaustively studied. Recent study has shown that microglia/macrophages could also clear myelin debris by phagocytosis, which creates a conducive environment for the regeneration of myelin. 44 It seems likely that ATL-III has multiple biological functions worth exploring in the future.
In conclusion, this study reveals a novel function for ATL-III in the regulation of microglial polarization both in vivo and in vitro and provides initial evidence that ATL-III has potential therapeutic benefits in promoting histological and functional repair in rats after SCI.
This effect may be partly mediated via inhibiting of neuroinflammation induced by secondary injury through the NF-κB, JNK MAPK, p38 MAPK, and Akt pathways. Given the established safety of ATL-III, its protective effect in an animal model of SCI suggests that this compound may have clinical potential for human SCI.

ACK N OWLED G M ENT
This study was supported by grants from the National Natural

CO N FLI C T O F I NTE R E S T
The authors declare no competing financial interests.